Rationally-designed mutations to the thrA gene for enhanced lysine production in E. coli

ABSTRACT

The present disclosure relates to various different types of mutations in the thrA gene in  E. coli  leading to enhanced lysine production for, e.g., supplements and nutraceuticals.

RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No.63/027,246, filed 19 May 2020, entitled “Rationally-Designed Mutationsto the TRAA Gene for Enhanced Lysine Production in E. Coli”,incorporated by reference herein in its entirety.

FIELD OF THE INVENTION

The present disclosure relates to mutations in the thrA gene in E. colileading to enhanced lysine production.

BACKGROUND OF THE INVENTION

In the following discussion certain articles and methods will bedescribed for background and introductory purposes. Nothing containedherein is to be construed as an “admission” of prior art. Applicantexpressly reserves the right to demonstrate, where appropriate, that thearticles and methods referenced herein do not constitute prior art underthe applicable statutory provisions.

The amino acid lysine is an α-amino acid that is used in thebiosynthesis of proteins and is a metabolite of E. coli, S. cerevisiae,plants, humans and other mammals, as well as algae. Lysine contains anα-amino group, an α-carboxylic acid group, and has a chemical formula ofC6H14N202 One of nine essential amino acids in humans, lysine isrequired for growth and tissue repair and has a role as a micronutrient,a nutraceutical, an agricultural feed supplement, an anticonvulsant, aswell as a precursor for the production of peptides. Because of theseroles as, e.g., a supplement and nutraceutical, there has been a growingeffort to produce lysine on a large scale.

Accordingly, there is a need in the art for organisms that produceenhanced amounts of lysine where such organisms can be harnessed forlarge scale lysine production. The disclosed nucleic acid sequences fromE. coli satisfy this need.

SUMMARY OF THE INVENTION

This Summary is provided to introduce a selection of concepts in asimplified form that are further described below in the DetailedDescription. This Summary is not intended to identify key or essentialfeatures of the claimed subject matter, nor is it intended to be used tolimit the scope of the claimed subject matter. Other features, details,utilities, and advantages of the claimed subject matter will be apparentfrom the following written Detailed Description including those aspectsillustrated in the accompanying drawings and defined in the appendedclaims.

The present disclosure provides E. coli variants havingrationally-designed mutations in the thrA gene that produce enhancedamounts of lysine in culture. Thus, in some embodiments, the presentdisclosure provides a thrA variant comprising a variant amino acidsequence comprising any one of SEQ ID NOs. 1-40, or any combinations ofvariant amino acid sequences SEQ ID NOs: 1-40; i.e., variant sequencesA660R, A720W, A840T, A808L, A080M, D648W, D763H, E649D, 5695G, E702R,F709W, G472N, G650F, G679P, G650P, G610H, G610K, G634I, G634K, I616P,I685H, I685K, K681F, K682G, K682I, K682Q, K682R, K772P, L631D, L631G,L768F, L811R, P467H, Q487D, Q487D, R661V, R668S, T8021, V468A and V468Y;or any combination of other variant sequences with any one or more ofvariant sequences A660R, A720W, A840T, A808L, A080M, D648W, D763H,E649D, 5695G, E702R, F709W, G472N, G650F, G679P, G650P, G610H, G610K,G634I, G634K, I616P, I685H, I685K, K681F, K682G, K682I, K682Q, K682R,K772P, L631D, L631G, L768F, L811R, P467H, Q487D, Q487D, R661V, R668S,T8021, V468A and V468Y.

These aspects and other features and advantages of the invention aredescribed below in more detail.

BRIEF DESCRIPTION OF THE DRAWINGS

The foregoing and other features and advantages of the present inventionwill be more fully understood from the following detailed description ofillustrative embodiments taken in conjunction with the accompanyingdrawings in which:

FIG. 1A is an exemplary engine vector for creating edits in E. coli.FIG. 1B is an exemplary editing vector for creating edits in E. coli.

It should be understood that the drawings are not necessarily to scale,and that like reference numbers refer to like features.

DETAILED DESCRIPTION

All of the functionalities described in connection with one embodimentof the methods, devices or instruments described herein are intended tobe applicable to the additional embodiments of the methods, devices andinstruments described herein except where expressly stated or where thefeature or function is incompatible with the additional embodiments. Forexample, where a given feature or function is expressly described inconnection with one embodiment but not expressly mentioned in connectionwith an alternative embodiment, it should be understood that the featureor function may be deployed, utilized, or implemented in connection withthe alternative embodiment unless the feature or function isincompatible with the alternative embodiment.

The practice of the techniques described herein may employ, unlessotherwise indicated, conventional techniques and descriptions molecularbiology (including recombinant techniques), cell biology, biochemistry,and genetic engineering technology, which are within the skill of thosewho practice in the art. Such conventional techniques and descriptionscan be found in standard laboratory manuals such as Green and Sambrook,Molecular Cloning: A Laboratory Manual. 4th, ed., Cold Spring HarborLaboratory Press, Cold Spring Harbor, N.Y., (2014); Current Protocols inMolecular Biology, Ausubel, et al. eds., (2017); Neumann, et al.,Electroporation and Electrofusion in Cell Biology, Plenum Press, NewYork, 1989; and Chang, et al., Guide to Electroporation andElectrofusion, Academic Press, California (1992), all of which areherein incorporated in their entirety by reference for all purposes.

Note that as used herein and in the appended claims, the singular forms“a,” “an,” and “the” include plural referents unless the context clearlydictates otherwise. Thus, for example, reference to “a cell” refers toone or more cells, and reference to “the system” includes reference toequivalent steps, methods and devices known to those skilled in the art,and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. All publications mentionedherein are incorporated by reference for the purpose of describing anddisclosing devices, formulations and methodologies that may be used inconnection with the presently described invention.

Where a range of values is provided, it is understood that eachintervening value, between the upper and lower limit of that range andany other stated or intervening value in that stated range isencompassed within the invention. The upper and lower limits of thesesmaller ranges may independently be included in smaller ranges, and arealso encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

In the following description, numerous specific details are set forth toprovide a more thorough understanding of the present invention. However,it will be apparent to one of skill in the art that the presentinvention may be practiced without one or more of these specificdetails. In other instances, features and procedures well known to thoseskilled in the art have not been described in order to avoid obscuringthe invention. The terms used herein are intended to have the plain andordinary meaning as understood by those of ordinary skill in the art.

The term DNA “control sequences” refers collectively to promotersequences, polyadenylation signals, transcription termination sequences,upstream regulatory domains, origins of replication, internal ribosomeentry sites, nuclear localization sequences, enhancers, and the like,which collectively provide for the replication, transcription andtranslation of a coding sequence in a recipient cell. Not all of thesetypes of control sequences need to be present so long as a selectedcoding sequence is capable of being replicated, transcribed and—for somecomponents—translated in an appropriate host cell.

The term “CREATE cassette” or “editing cassette” refers to a gRNA linkedto a donor DNA or HA. Methods and compositions for designing andsynthesizing CREATE editing cassettes are described in U.S. Pat. Nos.10,240,167; 10,266,849; 9,982,278; 10,351,877; 10,364,442; 10,435,715;and 10,465,207; and U.S. Ser. No. 16/550,092, filed 23 Aug. 2019; U.S.Ser. No. 16/551,517, filed 26 Aug. 2019; U.S. Ser. No. 16/773,618, filed27 Jan. 2020; and U.S. Ser. No. 16/773,712, filed 27 Jan. 2020, all ofwhich are incorporated by reference herein in their entirety.

As used herein the term “donor DNA” or “donor nucleic acid” refers tonucleic acid that is designed to introduce a DNA sequence modification(insertion, deletion, substitution) into a locus (e.g., a target genomicDNA sequence or cellular target sequence) by homologous recombinationusing nucleic acid-guided nucleases. For homology-directed repair, thedonor DNA must have sufficient homology to the regions flanking the “cutsite” or site to be edited in the genomic target sequence. The length ofthe homology arm(s) will depend on, e.g., the type and size of themodification being made. In many instances and preferably, the donor DNAwill have two regions of sequence homology (e.g., two homology arms) tothe genomic target locus. Preferably, an “insert” region or “DNAsequence modification” region—the nucleic acid modification that onedesires to be introduced into a genome target locus in a cell—will belocated between two regions of homology. The DNA sequence modificationmay change one or more bases of the target genomic DNA sequence at onespecific site or multiple specific sites. A change may include changing1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 35, 40, 50, 75, 100, 150, 200, 300,400, or 500 or more base pairs of the genomic target sequence. Adeletion or insertion may be a deletion or insertion of 1, 2, 3, 4, 5,10, 15, 20, 25, 30, 40, 50, 75, 100, 150, 200, 300, 400, or 500 or morebase pairs of the genomic target sequence.

The terms “guide nucleic acid” or “guide RNA” or “gRNA” refer to apolynucleotide comprising 1) a guide sequence capable of hybridizing toa genomic target locus, and 2) a scaffold sequence capable ofinteracting or complexing with a nucleic acid-guided nuclease.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or, more often in the context of the presentdisclosure, between two nucleic acid molecules. The term “homologousregion” or “homology arm” refers to a region on the donor DNA with acertain degree of homology with the target genomic DNA sequence.Homology can be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When a position in thecompared sequence is occupied by the same base or amino acid, then themolecules are homologous at that position. A degree of homology betweensequences is a function of the number of matching or homologouspositions shared by the sequences.

“Operably linked” refers to an arrangement of elements where thecomponents so described are configured so as to perform their usualfunction. Thus, control sequences operably linked to a coding sequenceare capable of effecting the transcription, and in some cases, thetranslation, of a coding sequence. The control sequences need not becontiguous with the coding sequence so long as they function to directthe expression of the coding sequence. Thus, for example, interveninguntranslated yet transcribed sequences can be present between a promotersequence and the coding sequence and the promoter sequence can still beconsidered “operably linked” to the coding sequence. In fact, suchsequences need not reside on the same contiguous DNA molecule (i.e.chromosome) and may still have interactions resulting in alteredregulation.

As used herein, the terms “protein” and “polypeptide” are usedinterchangeably. Proteins may or may not be made up entirely of aminoacids.

A “promoter” or “promoter sequence” is a DNA regulatory region capableof binding RNA polymerase and initiating transcription of apolynucleotide or polypeptide coding sequence such as messenger RNA,ribosomal RNA, small nuclear or nucleolar RNA, guide RNA, or any kind ofRNA transcribed by any class of any RNA polymerase I, II or III.Promoters may be constitutive or inducible, and in some embodiments thetranscription of at least one component of the nucleic acid-guidednuclease editing system is—and often at least three components of thenucleic acid-guided nuclease editing system are—under the control of aninducible promoter. A number of gene regulation control systems havebeen developed for the controlled expression of genes in plant, microbe,and animal cells, including mammalian cells, including the pL promoter(induced by heat inactivation of the CI857 repressor), the pPhIFpromoter (induced by the addition of 2,4 diacetylphloroglucinol (DAPG)),the pBAD promoter (induced by the addition of arabinose to the cellgrowth medium), and the rhamnose inducible promoter (induced by theaddition of rhamnose to the cell growth medium). Other systems includethe tetracycline-controlled transcriptional activation system(Tet-On/Tet-Off, Clontech, Inc. (Palo Alto, Calif.); Bujard and Gossen,PNAS, 89(12):5547-5551 (1992)), the Lac Switch Inducible system(Wyborski et al., Environ Mol Mutagen, 28(4):447-58 (1996); DuCoeur etal., Strategies 5(3):70-72 (1992); U.S. Pat. No. 4,833,080), theecdysone-inducible gene expression system (No et al., PNAS,93(8):3346-3351 (1996)), the cumate gene-switch system (Mullick et al.,BMC Biotechnology, 6:43 (2006)), and the tamoxifen-inducible geneexpression (Zhang et al., Nucleic Acids Research, 24:543-548 (1996)) aswell as others.

As used herein the term “selectable marker” refers to a gene introducedinto a cell, which confers a trait suitable for artificial selection.General use selectable markers are well-known to those of ordinary skillin the art. Drug selectable markers such as ampicillin/carbenicillin,kanamycin, nourseothricin N-acetyl transferase, chloramphenicol,erythromycin, tetracycline, gentamicin, bleomycin, streptomycin,rifampicin, puromycin, hygromycin, blasticidin, and G418 may beemployed. In other embodiments, selectable markers include, but are notlimited to sugars such as rhamnose. “Selective medium” as used hereinrefers to cell growth medium to which has been added a chemical compoundor biological moiety that selects for or against selectable markers.

The term “specifically binds” as used herein includes an interactionbetween two molecules, e.g., an engineered peptide antigen and a bindingtarget, with a binding affinity represented by a dissociation constantof about 10⁻⁷ M, about 10⁻⁸ M, about 10⁻⁹ M, about 10⁻¹⁰ M, about10⁻¹¹M, about 10⁻¹²M, about 10⁻¹³M, about 10⁻¹⁴M or about 10⁻¹⁵ M.

The terms “target genomic DNA sequence”, “cellular target sequence”, or“genomic target locus” refer to any locus in vitro or in vivo, or in anucleic acid (e.g., genome) of a cell or population of cells, in which achange of at least one nucleotide is desired using a nucleic acid-guidednuclease editing system. The cellular target sequence can be a genomiclocus or extrachromosomal locus.

The term “variant” may refer to a polypeptide or polynucleotide thatdiffers from a reference polypeptide or polynucleotide but retainsessential properties. A typical variant of a polypeptide differs inamino acid sequence from another reference polypeptide. Generally,differences are limited so that the sequences of the referencepolypeptide and the variant are closely similar overall and, in manyregions, identical. A variant and reference polypeptide may differ inamino acid sequence by one or more modifications (e.g., substitutions,additions, and/or deletions). A variant of a polypeptide may be aconservatively modified variant. A substituted or inserted amino acidresidue may or may not be one encoded by the genetic code (e.g., anon-natural amino acid). A variant of a polypeptide may be naturallyoccurring, such as an allelic variant, or it may be a variant that isnot known to occur naturally.

A “vector” is any of a variety of nucleic acids that comprise a desiredsequence or sequences to be delivered to and/or expressed in a cell.Vectors are typically composed of DNA, although RNA vectors are alsoavailable. Vectors include, but are not limited to, plasmids, fosmids,phagemids, virus genomes, synthetic chromosomes, and the like. As usedherein, the phrase “engine vector” comprises a coding sequence for anuclease to be used in the nucleic acid-guided nuclease systems andmethods of the present disclosure. The engine vector also comprises inE. coli, the λ Red recombineering system or an equivalent thereto whichrepairs the double-stranded breaks resulting from the cut by thenuclease. Engine vectors also typically comprise a selectable marker. Asused herein the phrase “editing vector” comprises a donor nucleic acid,optionally including an alteration to the cellular target sequence thatprevents nuclease binding at a PAM or spacer in the cellular targetsequence after editing has taken place, and a coding sequence for agRNA. The editing vector may also and preferably does comprise aselectable marker and/or a barcode. In some embodiments, the enginevector and editing vector may be combined; that is, all editing andselection components may be found on a single vector. Further, theengine and editing vectors comprise control sequences operably linkedto, e.g., the nuclease coding sequence, recombineering system codingsequences (if present), donor nucleic acid, guide nucleic acid(s), andselectable marker(s).

Library Design Strategy and Nuclease-Directed Genome Editing

Lysine is naturally synthesized in E. coli along the diaminopimelate(DAP) biosynthetic pathway. The thrA gene in E. coli is not directlyinvolved in the biosynthetic pathway; instead, the thrA has twoenzymatic activities required for the biosynthesis of threonine,aspartokinase I and homoserine dehydrogenase I. The thrA gene was clonedin a bacterial plasmid and its complete nucleotide sequence wasestablished. It contains 2460 base pairs that encode for a polypeptidechain of 820 amino acids. The gene contains an internal sequence thatresembles the structure of bacterial ribosome-binding sites, with an AUGpreceded by four triplets, each of which can be converted to a nonsensecodon by a single mutation, suggesting that the single polypeptide chainwas formed by the fusion of two genes and that initiation of translationmay occur inside the gene to give a protein fragment having only thehomoserine dehydrogenase activity.

Strain engineering strategies for increasing lysine production in E.coli and other industrially-relevant production hosts such asCorynebacterium glutamicum have historically focused on the genes in theDAP pathway as obvious targets for mutagenesis and over-expression.Beyond this short list of genes encoding the lysine biosyntheticenzymes, it is likely that additional loci throughout the E. coli genomemay also contribute appreciably (if less directly) to improved lysineyields in an industrial production setting. For this reason, targetedmutagenesis strategies which enable a broader query of the entire genomeare also of significant value to the lysine metabolic engineer.

The variants presented in this disclosure are the result of nucleicacid-guided nuclease editing of 370+ loci in the thrA gene in the MG1655strain of E. coli harboring an engine plasmid such as that shown in FIG.1A (such transformed MG1655 strain is referred to herein as E. colistrain EC83). The mutations included amino acid swaps, prematureterminations via stop codons and mutations to the thrA promoter. Theengine plasmid comprises a coding sequence for the MAD7 nuclease underthe control of the inducible pL promoter, the λ Red operonrecombineering system under the control of the inducible pBAD promoter(inducible by the addition of arabinose in the cell growth medium), thec1857 gene under the control of a constitutive promoter, as well as aselection marker and an origin of replication. As described above, the λRed recombineering system repairs the double-stranded breaks resultingfrom the cut by the MAD7 nuclease. The c1857 gene at 30° C. activelyrepresses the pL promoter (which drives the expression of the MAD7nuclease and the editing or CREATE cassette on the editing cassette suchas the exemplary editing vector shown in FIG. 1B); however, at 42° C.,the c1857 repressor gene unfolds or degrades, and in this state thec1857 repressor protein can no longer repress the pL promoter leading toactive transcription of the coding sequence for the MAD7 nuclease andthe editing (e.g., CREATE) cassette.

FIG. 1B depicts an exemplary editing plasmid comprising the editing(e.g., CREATE) cassette (crRNA, spacer and HA) driven by a pL promoter,a selection marker, and an origin of replication.

The 370+ nucleic acid mutations or edits made were generated using MAD7,along with a gRNA and donor DNA. A nucleic acid-guided nuclease such asMAD7 is complexed with an appropriate synthetic guide nucleic acid in acell and can cut the genome of the cell at a desired location. The guidenucleic acid helps the nucleic acid-guided nuclease recognize and cutthe DNA at a specific target sequence. By manipulating the nucleotidesequence of the guide nucleic acid, the nucleic acid-guided nuclease maybe programmed to target any DNA sequence for cleavage as long as anappropriate protospacer adjacent motif (PAM) is nearby. In certainaspects, the nucleic acid-guided nuclease editing system may use twoseparate guide nucleic acid molecules that combine to function as aguide nucleic acid, e.g., a CRISPR RNA (crRNA) and trans-activatingCRISPR RNA (tracrRNA). In other aspects, the guide nucleic acid may be asingle guide nucleic acid that includes both the crRNA and tracrRNAsequences.

A guide nucleic acid comprises a guide sequence, where the guidesequence is a polynucleotide sequence having sufficient complementaritywith a target sequence to hybridize with the target sequence and directsequence-specific binding of a complexed nucleic acid-guided nuclease tothe target sequence. The degree of complementarity between a guidesequence and the corresponding target sequence, when optimally alignedusing a suitable alignment algorithm, is about or more than about 50%,60%, 75%, 80%, 85%, 90%, 95%, 97.5%, 99%, or more. Optimal alignment maybe determined with the use of any suitable algorithm for aligningsequences. In some embodiments, a guide sequence is about or more thanabout 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25,26, 27, 28, 29, 30, 35, 40, 45, 50, 75, or more nucleotides in length.In some embodiments, a guide sequence is less than about 75, 50, 45, 40,35, 30, 25, 20 nucleotides in length. Preferably the guide sequence is10-30 or 15-20 nucleotides long, or 15, 16, 17, 18, 19, or 20nucleotides in length.

In the methods to generate the 370+ thrA mutations, the guide nucleicacids were provided as a sequence to be expressed from a plasmid orvector comprising both the guide sequence and the scaffold sequence as asingle transcript under the control of an inducible promoter. The guidenucleic acids are engineered to target a desired target sequence byaltering the guide sequence so that the guide sequence is complementaryto a desired target sequence, thereby allowing hybridization between theguide sequence and the target sequence. In general, to generate an editin the target sequence, the gRNA/nuclease complex binds to a targetsequence as determined by the guide RNA, and the nuclease recognizes aprotospacer adjacent motif (PAM) sequence adjacent to the targetsequence. The target sequences for the genome-wide mutagenesis hereencompassed 370+ loci throughout the thrA gene in the E. coli genome.

The guide nucleic acid may be and in the processes generating thevariants reported herein were part of an editing cassette that alsoencoded the donor nucleic acid. The target sequences are associated witha proto-spacer mutation (PAM), which is a short nucleotide sequencerecognized by the gRNA/nuclease complex. The precise preferred PAMsequence and length requirements for different nucleic acid-guidednucleases vary; however, PAMs typically are 2-7 base-pair sequencesadjacent or in proximity to the target sequence and, depending on thenuclease, can be 5′ or 3′ to the target sequence.

In certain embodiments, the genome editing of a cellular target sequenceboth introduces the desired DNA change to the cellular target sequenceand removes, mutates, or renders inactive a proto-spacer mutation (PAM)region in the cellular target sequence. Rendering the PAM at thecellular target sequence inactive precludes additional editing of thecell genome at that cellular target sequence, e.g., upon subsequentexposure to a nucleic acid-guided nuclease complexed with a syntheticguide nucleic acid in later rounds of editing. Thus, cells having thedesired cellular target sequence edit and an altered PAM can be selectedfor by using a nucleic acid-guided nuclease complexed with a syntheticguide nucleic acid complementary to the cellular target sequence. Cellsthat did not undergo the first editing event will be cut rendering adouble-stranded DNA break, and thus will not continue to be viable. Thecells containing the desired cellular target sequence edit and PAMalteration will not be cut, as these edited cells no longer contain thenecessary PAM site and will continue to grow and propagate.

As for the nuclease component of the nucleic acid-guided nucleaseediting system, a polynucleotide sequence encoding the nucleicacid-guided nuclease can be codon optimized for expression in particularcell types, such as archaeal, prokaryotic or eukaryotic cells. Thechoice of nucleic acid-guided nuclease to be employed depends on manyfactors, such as what type of edit is to be made in the target sequenceand whether an appropriate PAM is located close to the desired targetsequence. Nucleases of use in the methods described herein include butare not limited to Cas 9, Cas 12/CpfI, MAD2, or MAD7 or other MADzymes.As with the guide nucleic acid, the nuclease is encoded by a DNAsequence on a vector (e.g., the engine vector—see FIG. 1A) and be underthe control of an inducible promoter. In some embodiments—such as in themethods described herein—the inducible promoter may be separate from butthe same as the inducible promoter controlling transcription of theguide nucleic acid; that is, a separate inducible promoter drives thetranscription of the nuclease and guide nucleic acid sequences but thetwo inducible promoters may be the same type of inducible promoter(e.g., both are pL promoters). Alternatively, the inducible promotercontrolling expression of the nuclease may be different from theinducible promoter controlling transcription of the guide nucleic acid;that is, e.g., the nuclease may be under the control of the pBADinducible promoter, and the guide nucleic acid may be under the controlof the pL inducible promoter.

Another component of the nucleic acid-guided nuclease system is thedonor nucleic acid comprising homology to the cellular target sequence.In some embodiments, the donor nucleic acid is on the samepolynucleotide (e.g., editing vector or editing cassette) as the guidenucleic acid. The donor nucleic acid is designed to serve as a templatefor homologous recombination with a cellular target sequence nicked orcleaved by the nucleic acid-guided nuclease as a part of thegRNA/nuclease complex. A donor nucleic acid polynucleotide may be of anysuitable length, such as about or more than about 20, 25, 50, 75, 100,150, 200, 500, or 1000 nucleotides in length. In certain preferredaspects, the donor nucleic acid can be provided as an oligonucleotide ofbetween 20-300 nucleotides, more preferably between 50-250 nucleotides.The donor nucleic acid comprises a region that is complementary to aportion of the cellular target sequence (e.g., a homology arm). Whenoptimally aligned, the donor nucleic acid overlaps with (iscomplementary to) the cellular target sequence by, e.g., about 20, 25,30, 35, 40, 50, 60, 70, 80, 90 or more nucleotides. In many embodiments,the donor nucleic acid comprises two homology arms (regionscomplementary to the cellular target sequence) flanking the mutation ordifference between the donor nucleic acid and the cellular targetsequence. The donor nucleic acid comprises at least one mutation oralteration compared to the cellular target sequence, such as aninsertion, deletion, modification, or any combination thereof comparedto the cellular target sequence. Various types of edits were introducedherein, including site-directed mutagenesis, saturation mutagenesis,promoter swaps and ladders, knock-in and knock-out edits, SNP or shorttandem repeat swaps, and start/stop codon exchanges.

In addition to the donor nucleic acid, an editing cassette may compriseone or more primer sites. The primer sites can be used to amplify theediting cassette by using oligonucleotide primers; for example, if theprimer sites flank one or more of the other components of the editingcassette. In addition, the editing cassette may comprise a barcode. Abarcode is a unique DNA sequence that corresponds to the donor DNAsequence such that the barcode can identify the edit made to thecorresponding cellular target sequence. The barcode typically comprisesfour or more nucleotides. In some embodiments, the editing cassettescomprise a collection or library gRNAs and of donor nucleic acidsrepresenting, e.g., gene-wide or genome-wide libraries of gRNAs anddonor nucleic acids. The library of editing cassettes is cloned intovector backbones where, e.g., each different donor nucleic acid isassociated with a different barcode.

Variants of interest from the 370+ variants created and assayed with atleast 900-fold improvement over wildtype (MG1655) in minimal media arethose listed in Table 1 below. The thrA gene has an NCBI Gene ID of945803 and the edit is in relation to the amino acid residue of theamino acid sequence.

TABLE 1 Variants Phenotype, FOWT Phenotype, FIOPC (MG1655, (dapA E84T,SEQ ID No. Edit min medium) min medium SEQ ID No. 1 A660R 932.018 1.864SEQ ID No. 2 A720W 2039.723 4.079 SEQ ID No. 3 A804T 1148.346 2.297 SEQID No. 4 A808L 3034.660 6.069 SEQ ID No. 5 A080M 1490.789 2.982 SEQ IDNo. 6 D648W 2464.749 4.929 SEQ ID No. 7 D763H 2862.677 5.725 SEQ ID No.8 E649D 1853.637 3.707 SEQ ID No. 9 E695G 1583.835 3.168 SEQ ID No. 10E702R 2186.557 4.373 SEQ ID No. 11 F709W 1658.502 3.317 SEQ ID No. 12G472N 2233.949 4.468 SEQ ID No. 13 G650F 1161.607 2.323 SEQ ID No. 14G679P 1599.642 3.199 SEQ ID No. 15 G650P 2197.597 4.395 SEQ ID No. 16G610H 2958.276 5.917 SEQ ID No. 17 G610K 4823.148 9.646 SEQ ID No. 18G634I 1818.493 3.637 SEQ ID No. 19 G634K 2313.266 4.627 SEQ ID No. 20I616P 1768.396 3.537 SEQ ID No. 21 I685H 1385.364 2.771 SEQ ID No. 22I685K 2522.543 5.045 SEQ ID No. 23 K681F 1368.820 2.738 SEQ ID No. 24K682G 2188.667 4.377 SEQ ID No. 25 K682I 2258.957 4.518 SEQ ID No. 26K682Q 2497.239 4.994 SEQ ID No. 27 K682R 3976.188 7.952 SEQ ID No. 28K772P 1455.380 2.911 SEQ ID No. 29 L631D 2457.977 4.916 SEQ ID No. 30L631G 1053.649 2.107 SEQ ID No. 31 L768F 2534.033 5.068 SEQ ID No. 32L811R 1059.535 2.119 SEQ ID No. 33 P467H 1491.993 2.984 SEQ ID No. 34Q487D 2078.026 4.156 SEQ ID No. 35 Q487F 2085.004 4.170 SEQ ID No. 36R661V 1399.696 2.799 SEQ ID No. 37 R668S 1118.189 2.236 SEQ ID No. 38T802I 2069.932 4.140 SEQ ID No. 39 V468A 1192.620 2.385 SEQ ID No. 40V468Y 1481.573 2.963 In Table 1, “FOWT” is fold over wild type inminimal medium (FOWT MC1655 (minimal medium), “FIOPC” is fold improvedover positive control (MG1655, dapA E84T).

EXAMPLES

Mutagenesis libraries specifically targeting the thrA gene. All thrAvariants were screened at shallow sampling for lysine production viamass spec as described below.

Example 1: Editing Cassette and Backbone Amplification and Assembly

Editing Cassette Preparation: 5 nM oligonucleotides synthesized on achip were amplified using Q5 polymerase in 50 μL volumes. The PCRconditions were 95° C. for 1 minute; 8 rounds of 95° C. for 30seconds/60° C. for 30 seconds/72° C. for 2.5 minutes; with a final holdat 72° C. for 5 minutes. Following amplification, the PCR products weresubjected to SPRI cleanup, where 30 μL SPRI mix was added to the 50 μLPCR reactions and incubated for 2 minutes. The tubes were subjected to amagnetic field for 2 minutes, the liquid was removed, and the beads werewashed 2× with 80% ethanol, allowing 1 minute between washes. After thefinal wash, the beads were allowed to dry for 2 minutes, 50 μL 0.5× TEpH 8.0 was added to the tubes, and the beads were vortexed to mix. Theslurry was incubated at room temperature for 2 minutes, then subjectedto the magnetic field for 2 minutes. The eluate was removed and the DNAquantified.

Following quantification, a second amplification procedure was carriedout using a dilution of the eluate from the SPRI cleanup. PCR wasperformed under the following conditions: 95° C. for 1 minute; 18 roundsof 95° C. for 30 seconds/72° C. for 2.5 minutes; with a final hold at72° C. for 5 minutes. Amplicons were checked on a 2% agarose gel andpools with the cleanest output(s) were identified. Amplificationproducts appearing to have heterodimers or chimeras were not used.

Backbone Preparation: A 10-fold serial dilution series of purifiedbackbone was performed, and each of the diluted backbone series wasamplified under the following conditions: 95° C. for 1 minute; then 30rounds of 95° C. for 30 seconds/60° C. for 1.5 minutes/72° C. for 2.5minutes; with a final hold at 72° C. for 5 minutes. After amplification,the amplified backbone was subjected to SPRI cleanup as described abovein relation to the cassettes. The backbone was eluted into 100 μL ddH₂Oand quantified before nucleic acid assembly.

Isothermal Nucleic Acid Assembly: 150 ng backbone DNA was combined with100 ng cassette DNA. An equal volume of 2× Gibson Master Mix was added,and the reaction was incubated for 45 minutes at 50° C. After assembly,the assembled backbone and cassettes were subjected to SPRI cleanup, asdescribed above.

Example 2: Transformation of Editing Vector Library into E Cloni®

Transformation: 20 μL of the prepared editing vector Gibson Assemblyreaction was added to 30 μL chilled water along with 10 μL E Cloni®(Lucigen, Middleton, Wis.) supreme competent cells. An aliquot of thetransformed cells were spot plated to check the transformationefficiency, where >100× coverage was required to continue. Thetransformed E Cloni® cells were outgrown in 25 mL SOB+100 μg/mLcarbenicillin (carb). Glycerol stocks were generated from the saturatedculture by adding 500 μL 50% glycerol to 1000 μL saturated overnightculture. The stocks were frozen at −80° C. This step is optional,providing a ready stock of the cloned editing library. Alternatively,Gibson or another assembly of the editing cassettes and the vectorbackbone can be performed before each editing experiment.

Example 3: Creation of New Cell Line Transformed With Engine Vector

Transformation: 1 μL of the engine vector DNA (comprising a codingsequence for MAD7 nuclease under the control of the pL induciblepromoter, a chloramphenicol resistance gene, and the λ Redrecombineering system) was added to 50 μL EC83 strain E. coli cells. Thetransformed cells were plated on LB plates with 25 μg/mL chloramphenicol(chlor) and incubated overnight to accumulate clonal isolates. The nextday, a colony was picked, grown overnight in LB+25 μg/mL chlor, andglycerol stocks were prepared from the saturated overnight culture byadding 500 μL 50% glycerol to 1000 μL culture. The stocks of EC1comprising the engine vector were frozen at −80° C.

Example 4: Preparation of Competent Cells

A 1 mL aliquot of a freshly-grown overnight culture of EC83 cellstransformed with the engine vector was added to a 250 mL flaskcontaining 100 mL LB/SOB+25 μg/mL chlor medium. The cells were grown to0.4-0.7 OD, and cell growth was halted by transferring the culture toice for 10 minutes. The cells were pelleted at 8000×g in a JA-18 rotorfor 5 minutes, washed 3× with 50 mL ice cold ddH₂O or 10% glycerol, andpelleted at 8000× g in JA-18 rotor for 5 minutes. The washed cells wereresuspended in 5 mL ice cold 10% glycerol and aliquoted into 200 μLportions. Optionally at this point the glycerol stocks could be storedat −80° C. for later use.

Example 5: Screening of Edited Libraries for Lysine Production

Library stocks were diluted and plated onto 245×245 mm LB agar plates(Teknova) containing 100 μg/mL carbenicillin (Teknova) and 25 μg/mLchloramphenicol (Teknova) using sterile glass beads. Libraries werediluted an appropriate amount to yield ˜2000-3000 colonies on theplates. Plates were incubated ˜16 h at 30° C. and then stored at 4° C.until use. Colonies were picked using a QPix™ 420 (Molecular Devices)and deposited into sterile 1.2 mL square 96-well plates (ThomasScientific) containing 300 μL of overnight growth medium (EZ RichDefined Medium, w/o lysine (Teknova), 100 μg/mL carbenicillin and 25μg/mL chloramphenicol). Plates were sealed (AirPore sheets (Qiagen)) andincubated for ˜19 h in a shaker incubator (Climo-Shaker ISF1-X (Kuhner),30° C., 85% humidity, 250 rpm). Plate cultures were then diluted 20-fold(15 μL culture into 285 μL medium) into new 96-well plates containinglysine production medium (20 g/L ammonium sulfate (Teknova), 200 mM MOPSbuffer (Teknova), 3 mg/L Iron(II) sulfate heptahydrate (Sigma), 3 mg/LManganese (II) sulfate monohydrate (Sigma), 0.5 mg/L Biotin (Sigma), 1mg/L Thiamine hydrochloride (Sigma), 0.7 g/L Potassium chloride(Teknova), 20 g/L glucose (Teknova), 5 g/L Potassium phosphate monobasic(Sigma), 1 mL/L Trace metal mixture (Teknova), 1 mM Magnesium sulfate(Teknova), 100 μg/mL carbenicillin and 25 μg/mL chloramphenicol).Production plates were incubated for 24 h in a shaker incubator(Climo-Shaker ISF1-X (Kuhner), 30° C., 85% humidity, 250 rpm).

Production plates were centrifuged (Centrifuge 5920R, Eppendorf) at3,000 g for 10 min to pellet cells. The supernatants from productionplates were diluted 100-fold into water (5 μL of supernatant with 495μL) of water in 1.2 mL square 96-well plates. Samples were thoroughlymixed and then diluted a subsequent 10-fold further into a 50:50 mixtureof acetonitrile and water (20 μL sample with 180 μL of theacetonitrile/water mixture) into a 96-well Plate (polypropylene, 335μL/well, Conical Bottom (Thomas Scientific). Plates were heat sealed andthoroughly mixed.

Lysine concentrations were determined using a RapidFire high-throughputmass spectrometry system (Agilent) coupled to a 6470 Triple Quad massspectrometer (Agilent). The RapidFire conditions were as follows: Pump1: 80% acetonitrile (LC/MS grade, Fisher), 20% water (LC/MS grade,Fisher), 1.5 mL/min, Pump 2: 100% water, 1.25 mL/min, Pump 3: 5%acetonitrile, 95% water, 1.25 mL/min. RapidFire method: Aspirate: 600ms, Load/wash: 2000 ms, Extra wash: 0 ms, Elute: 3000 ms,Re-equilibrate: 500 ms. 10 μL injection loop.

Example 6: Mass Spectrometry Conditions for Lysine Detection

Precursor ion: 147.1 m/z, Product ion (quantifying): 84 m/z, Dwell: 20,Fragmentor: 80, Collision energy: 20, Cell accelerator voltage: 4,Polarity: positive Precursor ion: 147.1 m/z, Product ion (qualifying):130 m/z, Dwell: 20, Fragmentor: 80, Collision energy: 8, Cellaccelerator voltage: 4, Polarity: positive Source conditions: Gas Temp:300° C., Gas Flow: 10 L/min, Nebulizer: 45 psi, Sheath gas temp: 350°C., Sheath gas flow: 11 L/min, Capillary voltage: 3000V (positive),Nozzle voltage: 1500V (positive)

Data was analyzed using MassHunter Quantitative Analysis software(Agilent) with a standard curve of lysine used for quantitation oflysine in the samples. Each 96-well plate of samples contained 4replicates of the wildtype strain and 4 replicates of the dapA E84Tpositive control strain to calculate the relative lysine yield ofsamples compared to the controls. Hits from the primary screen werere-tested in quadruplicate using a similar protocol as described above.

While this invention is satisfied by embodiments in many differentforms, as described in detail in connection with preferred embodimentsof the invention, it is understood that the present disclosure is to beconsidered as exemplary of the principles of the invention and is notintended to limit the invention to the specific embodiments illustratedand described herein. Numerous variations may be made by persons skilledin the art without departure from the spirit of the invention. The scopeof the invention will be measured by the appended claims and theirequivalents. The abstract and the title are not to be construed aslimiting the scope of the present invention, as their purpose is toenable the appropriate authorities, as well as the general public, toquickly determine the general nature of the invention. In the claimsthat follow, unless the term “means” is used, none of the features orelements recited therein should be construed as means-plus-functionlimitations pursuant to 35 U.S.C. § 112, ¶6.

We claim:
 1. A modified E. coli cell comprising a mutated thrA geneencoding a polypeptide comprising an amino acid sequence selected fromthe group consisting of SEQ ID NO:1 to SEQ ID NO: 40, wherein themodified E. coli cell produces an enhanced amount of lysine incomparison to a wildtype cell.
 2. The modified E. coli cell of claim 1,wherein the mutated thrA gene encodes a polypeptide comprising the aminoacid sequence as set forth in SEQ ID NO:
 17. 3. The modified E. colicell of claim 1, wherein the E. coli cell is of E. coli strain MG1655.4. The modified E. coli cell of claim 1, wherein the mutated thrA geneis generated using a nucleic acid-guided nuclease.
 5. The modified E.coli cell of claim 1, wherein the modified cell further comprises aselectable marker.
 6. The modified E. coli cell of claim 1, wherein themodified cell further comprises a barcode DNA sequence.